INFLAMMATION-RESPONSIVE ANTI-INFLAMMATORY HYDROGELS
The present invention relates generally to the field of protease-responsive drug delivery hydrogels, uses thereof, and related methods of their production. More particularly, the invention relates to hydrogels which release anti-inflammatory agents upon reaction with inflammation-related proteases.
The present application is a filing under 35 U.S.C. 371 as the National Stage of International Application No. PCT/SG2020/050723, filed Dec. 7, 2020, entitled “INFLAMMATION-RESPONSIVE ANTI-INFLAMMATORY HYDROGELS,” which claims priority to Singapore Application No. SG 10201911767R filed with the Intellectual Property Office of Singapore on Dec. 6, 2019, both of which are incorporated herein by reference in their entirety for all purposes.
INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILEThis application incorporates by reference the Sequence Listing contained in the following ASCII text file being submitted concurrently herewith:
File name: 4373-17500_SP101987USZBD_Sequence_Listing; created on Jun. 3, 2022; and having a files size of 44 KB.
The information in the Sequence Listing is incorporated herein in its entirety for all purposes.
FIELD OF THE INVENTIONThe present invention relates generally to the field of protease-responsive drug delivery hydrogels, use thereof, and related methods of their production. More particularly, the invention relates to hydrogels which release anti-inflammatory agents upon reaction with inflammation-related proteases.
BACKGROUND OF THE INVENTIONInflammation is a sequence of biological reactions mounted by the host immune system to remove harmful stimuli and restore a damaged tissue to its pre-injury condition [Serhan, C. N. et al. Fundamentals of Inflammation, Cambridge University Press, Cambridge, (2010)]. An acute inflammatory response is essential to eliminate harmful stimuli and restore cellular homeostasis after tissue injury [Serhan, C. N. et al. Fundamentals of Inflammation, Cambridge University Press, Cambridge, (2010)]. In contrast, undesirable persistence of leukocyte activity results in excessive inflammation associated with chronic tissue damage [Serhan, C. N. et al. Fundamentals of Inflammation, Cambridge University Press, Cambridge, (2010)]. This chronic condition is often encountered in many pathological conditions such as rheumatoid arthritis, chronic diabetic ulcers, inflammatory bowel diseases (IBDs) and chronic obstructive pulmonary diseases [Serhan, C. N. et al. Fundamentals of Inflammation, Cambridge University Press, Cambridge, (2010)].
Systemic administration of anti-inflammatory therapeutics is a clinically accepted treatment paradigm to mitigate excessive inflammation in chronic diseases. Small molecule drugs such as nonsteroidal anti-inflammatory drugs (NSAIDs) and steroidal immuno-suppressants are empirically prescribed to patients with rheumatoid arthritis and IBDs based on their clinical symptoms. However, systemic administration of these drugs is also implicated in the occurrence of well-known side effects, which are associated with excessive dosage resulting from a lack of controlled drug release. For instance, systematically administered NSAIDs raise the risk of myocardial infarction, cerebrovascular accidents, and gastric ulceration. In addition, corticosteroids result in severe drug-induced complications such as osteonecrosis, glaucoma, and opportunistic infection when prescribed over an extended duration.
To improve spatiotemporal control of drug release kinetics and minimize systemic toxicity, several drug delivery platforms have been designed for administration of anti-inflammatory therapeutics [Hämäläinen, M. et al. Basic & Clinical Pharmacology & Toxicology 112(5) 296-301 (2013)]. For example, encapsulating glucocorticoids in vesicular systems extended drug half-life and achieved a slower release of therapeutic drugs [Maestrelli, F. et al. Journal of Drug Delivery Science and Technology 32 192-205 (2016)]. Alternatively, covalent conjugation of a small molecule NSAID to nanoscale polymeric films has been reported as an alternative approach to substantially increase therapeutic payload and achieve gradual long-term release by hydrolysis of the drug-polymer ester linkage [Hsu, B. B. Proceedings of the National Academy of Sciences 111(33) 12175 (2014)]. However, these systems do not take into account the specific pathological conditions of the diseased tissues whose inflammatory characteristics necessitate the administration of anti-inflammatory therapeutics. Therefore, their drug release profiles do not match the biological requirements as the release is primarily dictated by the physiochemical characteristics of the delivery platforms such as polymer composition and drug loading capacity.
The inflammatory characteristics of the biological microenvironment at diseased tissues can be leveraged to design smart drug delivery systems which can be triggered by immunological cues. Particularly, published studies have established the upregulation of proteases, especially serine proteases and matrix metalloproteases (MMPs), in chronic inflammation, suggesting their potential as biochemical cues for therapeutic administration to modulate the inflammation cascade [Pham, C. T. N. The International Journal of Biochemistry & Cell Biology 40(6) 1317-1333 (2008)]. Compared to other stimuli such as pH, temperature, or redox, proteases still stand as the more specific biological cue as compared to the other stimuli, mainly due to the close relationship between dysregulation of proteases with a pathological condition. Moreover, other stimuli can be affected due to environmental conditions. For instance, body temperature can spike due to the humid weather condition and not due to the disease state. Despite proteases being a better biological cue, the potential of proteases as immunological cues for biologically-triggered drug delivery systems to modulate inflammation has largely remained unexplored. Recently, Joshi et al. leveraged the self-assembly of a small molecule amphiphile, triglycerol monostearate (TG-18), to physically entrap a corticosteroid in a hydrogel platform which could be triggered to release this drug upon exposure to increased arthritic flare activity [Joshi, N. et al. Nature Communications 9(1) 1275 (2018)]. Nonetheless, this reported drug-loaded hydrogel platform lacks a generalizable design framework, thus limiting the possibility of replacing its components to exploit alternative biological triggers. Specifically, drug release from this platform relies on the cleavage of ester bonds on the TG-18 backbone primarily by esterases, which are upregulated in inflammatory arthritis [Ravaud, P. et al. Rheumatology 41(7) 815-818 (2002)] but might not be a critical biological cue in other inflammatory diseases. Non-enzymatic hydrolysis of these ester bonds in the low pH environment associated with inflammatory conditions [Bellocq, A., et al. Journal of Biological Chemistry 273(9) 5086-5092 (1998); Riemann, A. et al. Molecular Basis of Disease 1862(1) 72-81 (2016)] might also result in undesirable non-specific drug release.
Therefore, a protease-triggered drug delivery platform that is (1) modular in design, (2) immuno-compatible and (3) versatile for both injectable and topical administration at room temperature still represents an unmet need to address limitations of the existing delivery systems. Firstly, physical entrapment of a drug in a particulate domain such as liposome or polymeric microparticles embedded in a protease-triggered delivery systems might be associated with a diffusion-driven basal drug release. This basal release might be desirable for management of chronic inflammatory conditions that requires a protease-triggered increased dosage when the condition is suddenly exacerbated due to infection onset or arthritic flares. However, this basal release is not always desirable in all inflammation-associated conditions, particularly in immuno-compromised patient or patients on immuno-suppressant drugs or for management of acute injury where some extent of inflammation is required for normal healing. An alternative design of protease-triggered delivery system which eliminates or minimizing this basal drug release is also desirable for conditions in which the drug-administered site undergoes a transition from physiological state requiring no drug to highly inflammatory pathological states such as sudden onset of bacterial infection on acute wounds or flare-up of seborrheic dermatitis.
Secondly, leveraging a single protease as a biochemical stimulus for triggering drug release in the management of inflammation-associated pathology can partially help to tailor the dosage to the inflammation condition of the diseases. However, multiple proteases might be upregulated in a pathological inflammatory condition. Therefore, utilizing a subset of proteases instead of a single protease can increase the specific association of protease activity with disease-specific condition to achieve drug release kinetics specifically tailored to the inflammation-associated disease of interest. Thus, there also remains a substantial need for the development of a drug delivery system whose drug release is triggered by two or more protease stimuli (or plural protease responsivity) to achieve enhanced specificity.
SUMMARY OF THE INVENTIONThe present invention provides an inflammation-responsive drug delivery platform comprising of (1) drug-loaded domains (either particles encapsulating anti-inflammatory drugs or conjugated anti-inflammatory drug) with a tailored basal drug release profile and/or (2) a proteases-cleavable hydrogel domain. This invention provides a drug delivery platform which can be customized to cope with an inflammatory disease by changing the configuration of its drug-loaded domain and/or adjusting the plural sensitivity of its protease-triggered domain to tailor its responsiveness and specificity to the disease of interest.
According to a first aspect of the invention, there is provided a drug-loaded protease-responsive hydrogel comprising;
a) a drug encapsulated in particles;
b) a polymer building block comprising of a multi-arm-polyethylene glycol (PEG) with functional moiety; and
c) a bis-functional protease-sensitive crosslinker comprising a protease-cleavable substrate flanked by two spacer sequences containing functional moieties;
wherein said polymer building block of b) forms a gel in the presence of the protease-cleavable crosslinker of c) to entrap the particles of a).
In some embodiments the drug-loaded protease-responsive hydrogel further comprises:
a) at least a second bis-functional protease-sensitive crosslinker which comprises a protease-cleavable substrate, sensitive to a different protease to that of said crosslinker of c), flanked by spacer sequences containing functional moieties; and/or
b) at least a further bis-functional protease-resistant crosslinker which comprises a protease-resistant substrate.
The particles may be comprised of any suitable material that can carry and release a drug (such as a small molecule, therapeutic peptide, protein, mRNA, or the like) and be entrapped by the gel formed by the polymer building block and crosslinker. For example, the particles may be silica, liposomes, siRNA complexes or polymeric material. Particles can be made using well-known prior art methods such as emulsion, electrospraying, electrostatic complexation, flow-focusing method, etc., [Abdelaziza, Hadeer M. et al., Journal of Controlled Release 269 374-392 (2018)].
In some embodiments the drug is encapsulated in particles comprising a polymeric material selected from the group comprising polycaprolactone, poly (methacrylic acids), polylactic acids, polyvinylpirrolidone, poly(lactic-co-glycolic acid) (PLGA) and gelatin. Preferably, the particles are microparticles and/or nanoparticles, preferably having a diameter in the range of about 10 nm to about 100 μm.
In some embodiments the polymer building block comprises a multi-arm-PEG-vinyl sulfone or multi-arm-PEG-maleimide or multi-arm-PEG-azide or multi-arm-PEG-alkyne. Advantageously, the sulfone moiety interacts with a cysteine moiety on an arm of the crosslinker.
The invention also embodies a drug-loaded protease-responsive hydrogel that does not require encapsulation of the drug in particles for containment until released by said protease.
According to a second aspect of the invention, there is provided a drug-loaded protease-responsive hydrogel comprising;
a) a drug covalently conjugated to a protease-cleavable peptide anchor having a functional moiety;
b) a polymer building block comprising a multi-arm-PEG polymer having at least one functional moiety; and
c) a bis-functional crosslinker comprising a peptide substrate flanked by spacer sequences containing functional moieties;
wherein the functional moiety of the peptide anchor covalently links the drug to an arm of the multi-arm PEG polymer, and wherein a functional moiety of said polymer building block covalently links to said moiety of said bis-functional crosslinker to form a gel.
The arrangement of peptide anchor and crosslinker provides flexibility and tuning of the release profile of the drug-loaded hydrogel, whereby the release of the drug may be sensitive to one or more different proteases.
Advantageously, the drug-conjugated domain minimizes basal release of the drug.
In some embodiments:
a) said crosslinker is not cleavable to a protease; or
b) said peptide anchor is cleavable to a protease and said crosslinker is cleavable to the same or different protease; and/or
c) said drug-loaded hydrogel comprises a plurality of crosslinkers, one or more of which are cleavable to different proteases.
Advantageously, a desired peptide anchor consists of a protease-cleavable spacer sequence containing a functional moiety, which comprises at least 4 amino acids.
Advantageously, a crosslinker consists of a protease-cleavable substrate sequence flanked by spacer sequences containing functional moieties, each of which comprises at least 4 amino acids.
The non-cleavable crosslinker is used to control the diffusion of the enzyme into the gel network and hence help to tune the release profile.
In some embodiments the drug may be a small molecule, siRNA, aptamer or therapeutic peptide or protein.
Advantageously, the combination of peptide sequences, which are the key component of the protease-triggered domain, provide a fast water-based gelation and better specifically-triggered release upon exposure to more than one disease-specific proteases.
In some embodiments the polymer building block comprises a multi-arm-PEG-vinyl Maleimide. The amount of drug loaded onto the protease-responsive hydrogel may be controlled by the amount or concentration of multi-arm-PEG polymer used.
In some embodiments the weight ratio of the drug-loaded protease-responsive hydrogel is from about 2 w/v % to about 12 w/v %, preferably from about 3 w/v % to about 10 w/v %. Preferably the hydrogel is a multi-arm-PEG-vinyl sulfone or a multi-arm-PEG-vinyl Maleimide or a multi-arm-PEG-alkyne or a multi-arm-PEG-azide.
It would be understood that the number of arms on the multi-arm-PEG polymer will have an effect on the amount of drug that can be conjugated and also on the degree of crosslinking and gel formation.
In some embodiments of the drug-loaded protease-responsive hydrogel of any aspect of the invention, the multi-arm PEG polymer has 3 to 8 arms.
In some embodiments of the drug-loaded protease-responsive hydrogel of any aspect of the invention, the drug is anti-inflammatory.
In some embodiments of the drug-loaded protease-responsive hydrogel of any aspect of the invention, the protease is upregulated during inflammation and is selected from the group comprising matrix metalloproteinases and serine proteases.
In some embodiments of the drug-loaded protease-responsive hydrogel of any aspect of the invention, the drug is a steroidal anti-inflammatory drug or a non-steroidal anti-inflammatory drug (NSAID), or derivatives thereof. The drug may be a steroidal anti-inflammatory drug such as Dexamethasone, Fludrocortisone, Methylprednisolone, Prednisolone, Prednisone or Hydrocortisone, or derivatives thereof. Glucocorticoids can be oxidized to add a carboxylic functional group which allows these drugs to be conjugated to the peptide anchors of the invention. Preferably the drug is a NSAID, such as Ibuprofen, Ketoprofen, Diclorofenac, Sunlindac, Piroxicam, or Celecoxib, or derivatives thereof.
In some embodiments of the drug-loaded protease-responsive hydrogel of any aspect of the invention, the said flanking spacer sequences comprise at least one Cysteine and/or Lysine residue and/or azide- or alkyne-containing unnatural amino acid which are required to react with the functional moiety of the multi-arm PEGs to induce gelation. The spacer may have 1-6 amino acids. The remaining residues can be any of the amino acids, preferably amino acid with charged side groups. Specifically, positive charges amino acids (e.g., arginine, R) close to thiol moieties of cysteines increase the crosslinking rate while negative charges (e.g., aspartic acid, D) decelerated this reaction. The spacer may have 1-6 amino acids, In some embodiments, the flanking spacer sequence (“SPACER”) may be of the formula GX1X2X3, (SEQ ID NO: 33) wherein each of X1, X2 and X3 is independently Glycine, Cysteine, Aspartic acid, or Arginine and/or the reverse sequence thereof. In some embodiments, the said flanking spacer sequences are selected from the group comprising GRCR (SEQ ID NO; 1), GCRG (SEQ ID NO: 2), GRCD (SEQ ID NO: 3), GCDR (SEQ ID NO: 4), GCDG (SEQ ID NO: 5), GDCD (SEQ ID NO: 6), GCDD (SEQ ID NO: 7), GCRD (SEQ ID NO: 8) and GCRR (SEQ ID NO: 9).
When first and second spacers are used, one at each end of a peptide substrate, the second spacer sequence may be the reverse of the first spacer sequence and may be of the formula X3 X2 X1 G (SEQ ID NO: 34). This reversed spacer sequence may be referred to as a “RECAPS” and, for example, be the reverse sequence of a spacer selected from the group comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, ID NO: 8 and SEQ ID NO: 9.
In some embodiments of the drug-loaded protease-responsive hydrogel of any aspect of the invention the protease-cleavable substrate is sensitive to a protease selected from the group comprising matrix metalloproteinases, such as metalloproteinase-9 (MMP-9), MMP-2, MMP-7, MMP-12 etc., cathepsins, such as Cathepsin K, Cathepsin B, Cathepsin S, etc., human neutrophil elastase (HNE), caspases and urokinases.
In some embodiments, the protease-cleavable substrate is selected from the group comprising MMP-9 substrates comprising the amino acid sequence set forth in KGPRSLSGK (SEQ ID NO: 30), GPRSLSG (SEQ ID NO: 10), LGRMGLPGK (SEQ ID NO: 11), AVRWLLTA (SEQ ID NO: 12) or GPQGIWGQ (SEQ ID NO: 13); HNE substrates comprising APEEIMDRQ (SEQ ID NO: 14) or PMAVVQSVP (SEQ ID NO: 15); Cathepsin B substrates comprising GRRGLG (SEQ ID NO: 16) or DGFLGDD (SEQ ID NO: 17) or a combination thereof.
According to a third aspect of the invention, there is provided a composition comprising the drug-loaded protease-responsive hydrogel of any aspect of the invention formulated for injection or topical administration.
The drug-loaded inflammation-responsive hydrogel can be incorporated onto a polymeric dressing to form a composite dressing for wound management.
According to a fourth aspect of the invention, there is provided a dressing comprising the drug-loaded protease-responsive hydrogel of any aspect of the invention.
According to a fifth aspect of the invention, there is provided of the drug-loaded protease-responsive hydrogel of any aspect of the invention or a composition of the invention as an injectable or topical dressing for treating a subject in need thereof.
According to a sixth aspect of the invention, there is provided a method of treatment comprising administering to a subject in need of such treatment an efficacious amount of the drug-loaded protease-responsive hydrogel of any aspect of the invention or a composition of the invention. In some embodiments the administration is by injection or topical application to the subject. In some embodiments, the treatment is for inflammation-associated diseases such as chronic wounds, inflammatory bowel diseases, arthritis and potentially infection-related conditions for which inflammation management is desirable.
According to a seventh aspect of the invention, there is provided a kit comprising:
a) a drug encapsulated in particles;
b) a polymer building block comprising of a multi-arm-polyethylene glycol (PEG) with functional moiety; and
c) a bis-functional protease-sensitive crosslinker comprising a protease-cleavable substrate flanked by two spacer sequences containing functional moieties, wherein a)-c) are as defined in any one of the previous aspects; or
a) a drug covalently conjugated to a protease-cleavable peptide anchor having a functional moiety;
b) a polymer building block comprising a multi-arm-PEG polymer having at least one functional moiety; and
c) a bis-functional crosslinker comprising a peptide substrate flanked by spacer sequences containing functional moieties, wherein a)-c) are as defined in any one of the previous aspects.
In some embodiments, the kit comprises the drug-loaded protease-responsive hydrogel of any aspect of the invention or a composition of any aspect of the invention.
According to an eighth aspect of the invention, there is provided a method of manufacturing a drug-loaded protease-responsive hydrogel comprising the steps:
a) mixing a polymer building block comprising of a multi-arm-polyethylene glycol (PEG) with functional moiety with drug-loaded particles;
b) mixing a bis-functional protease-sensitive crosslinker, comprising a protease-cleavable substrate flanked by spacer sequences containing functional moieties, with drug-loaded polymeric particles;
c) mixing together the mixtures of a) and b);
wherein said polymer building block of a) forms a gel in the presence of the protease-cleavable crosslinker of b) to entrap the drug-loaded particles.
The particles may be comprised of any suitable material that can carry and release a drug (such as a small molecule, therapeutic peptide, protein, mRNA, or the like) and be entrapped by the gel formed by the polymer building block and crosslinker. For example, the particles may be silica, liposomes, siRNA complexes or polymeric material. Particles can be made using well-known prior art methods such as emulsion, electrospraying, electrostatic complexation, flow-focusing method, etc., [Abdelaziza, Hadeer M. et al., Journal of Controlled Release 269 374-392 (2018)].
In some embodiments the drug is encapsulated in particles comprising a polymeric material selected from the group comprising polycaprolactone, poly (methacrylic acids), polylactic acids, polyvinylpirrolidone, poly(lactic-co-glycolic acid) (PLGA) and gelatin. Preferably, the particles are microparticles and/or nanoparticles, preferably having a diameter in the range of about 10 nm to about 100 μm.
According to a ninth aspect of the invention, there is provided a method of manufacturing a drug-loaded protease-responsive hydrogel comprising the steps:
a) mixing a drug which is covalently conjugated to a peptide anchor having a functional moiety with a polymer building block comprising a multi-arm-PEG polymer having at least one functional moiety, wherein the respective functional moieties of the peptide anchor and multi-arm-PEG polymer covalently bond to conjugate the drug to an arm of the multi-arm PEG polymer;
b) mixing the drug-polymer conjugate of a) with a bis-functional crosslinker comprising a peptide substrate flanked by spacer sequences containing functional moieties;
wherein a functional moiety of said polymer building block covalently links to said moiety of said bis-functional crosslinker to form a gel.
In some embodiments of the ninth aspect:
a) said peptide anchor is cleavable to a protease and said crosslinker is not cleavable to a protease; or
b) said peptide anchor is cleavable to a protease and said crosslinker is cleavable to the same or different protease; and/or
c) said drug-loaded hydrogel comprises a plurality of crosslinkers, one or more of which are cleavable to different proteases.
In some embodiments, the drug, the particles, the crosslinker, the cleavable anchor and/or the polymer building block are as defined in any aspect of the invention.
According to a tenth aspect of the invention, there is provided a method of manufacturing a composite dressing comprising a drug-loaded protease-responsive hydrogel of any aspect of the invention, comprising the steps;
a) preparing a mixture of a drug-encapsulated in particles and a bis-functional protease-sensitive crosslinker comprising a protease-cleavable substrate flanked by two spacer sequences containing functional moieties;
b) preparing a mixture of a drug-encapsulated in particles and a polymer building block comprising of a multi-arm-polyethylene glycol (PEG) with functional moiety;
c) mixing a) and b) together, depositing the mixture onto a dressing and allowing gelation.
In some embodiments, the dressing is an alginate wound dressing.
In some embodiments, the method further comprises step d), wherein the composite dressing is flash-frozen in liquid nitrogen and lyophilized to dryness.
In some embodiments of the method of manufacturing, the drug is a NSAID; the particle comprises poly(lactic-co-glycolic acid) (PLGA); the crosslinker and/or anchor are cleavable to a protease selected from the group comprising matrix metalloproteinases and serine proteases or combinations thereof; and the polymer building block comprises a 4- or 8-arm-PEG-vinyl sulfone or a 4- or 8-arm-PEG-vinyl Maleimide or a 4 or 8-arm-PEG-azide or a 4 or 8-arm-PEG-alkyne.
Advantageously, the generalizable design framework enables the changes in choices and loading capacity of drugs while maintaining its structural and functional integrity.
Advantageously, immuno-compatible materials are utilized in the design of this delivery platform to potentially minimize adverse host response upon its administration in vivo.
Advantageously, this platform is versatile for both injectable and topical administration at room temperature.
Bibliographic references mentioned in the present specification are for convenience listed at the end of the examples. The whole content of such bibliographic references is herein incorporated by reference.
DETAILED DESCRIPTION OF THE INVENTION DefinitionsCertain terms employed in the specification, examples and appended claims are collected here for convenience.
As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
As used herein, ranges can be expressed as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10” as well as “greater than or equal to 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 3 and 10 are disclosed, then 4, 5, 6, 7, 8, and 9 are also disclosed.
The terms “amino acid” or “amino acid sequence,” as used herein, refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where “amino acid sequence” is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
As used herein, the terms “polypeptide”, “peptide” or “protein” refer to one or more chains of amino acids, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein said polypeptide or peptide can comprise a plurality of chains non-covalently and/or covalently linked together by peptide bonds, having the sequence of native proteins, that is, proteins produced by naturally-occurring and specifically non-recombinant cells, or genetically-engineered or recombinant cells, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence. A “polypeptide”, “peptide” or “protein” can comprise one (termed “a monomer”) or a plurality (termed “a multimer”) of amino acid chains. The term “particle” is used herein to broadly describe a material that encapsulates a drug and may be comprised of any suitable material that can carry and release the drug (such as a small molecule, therapeutic peptide, protein, mRNA, or the like) and be entrapped by the gel formed by the polymer building block and crosslinker. For example, the particles may be silica, liposomes, siRNA complexes or polymeric material. Particles can be made using well-known prior art methods such as emulsion, electrospraying, electrostatic complexation, flow-focusing method, etc., [Abdelaziza, Hadeer M. et al., Journal of Controlled Release 269 374-392 (2018)]. Polymeric particles are generally spheroidal in shape as shown in
The term “polymer” or “biopolymer” is defined as a substance with repeated molecular units to become polymeric. The polymer may be a biocompatible polymer, selected from the group comprising polysaccharide (e.g. agarose, dextran), polyphosphazene, poly(acrylic acids), poly(methacrylic acids), copolymers of acrylic acid and methacrylic acid, poly(alkylene oxidase), poly(vinyl acetate), polyvinylpyrrolidone (PVP), their derivatives and copolymers and blends thereof. In respect of drug-encapsulated polymeric particles, the polymer may be, for example, selected from the group comprising polycaprolactone, poly (methacrylic acids), polylactic acids, polyvinylpirrolidone, poly(lactic-co-glycolic acid) (PLGA) and gelatin. The polymer may be a flexible polymer that is also mechanically and structurally stable and suitable for injection, transplantation or implantation (e.g. subcutaneous transplantation or implantation). The polymer may or may not be biodegradable. Polymer building blocks of the invention generally comprise a plurality of arms which have functional moieties that can interact with a functional moiety on a crosslinker to form a gel. Preferred multi-arm building blocks include multi-arm-PEG-vinyl sulfone, multi-arm-PEG-vinyl Maleimide, multi-arm-PEG-zide and multi-arm-PEG-alkyne, more particularly those with 4 or 8 arms.
The term “subject” is herein defined as vertebrate, particularly mammal, more particularly human. For purposes of research, the subject may particularly be at least one animal model, e.g., a mouse, rat and the like. In particular, for treatment or prophylaxis of a disease, such as an inflammatory disease, the subject may be a human.
The term ‘treatment’, as used in the context of the invention refers to prophylactic, ameliorating, therapeutic or curative treatment.
As used herein, the term “comprising” or “including” is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. However, in context with the present disclosure, the term “comprising” or “including” also includes “consisting of”. The variations of the word “comprising”, such as “comprise” and “comprises”, and “including”, such as “include” and “includes”, have correspondingly varied meanings.
While aspects of the present invention will be described in conjunction with the embodiments provided herein, it will be understood that they are not intended to limit the present invention to these embodiments. On the contrary, the present invention is intended to cover alternatives, modifications and equivalents to the embodiments described herein, which are included within the scope of the present invention as defined by the appended claims. Furthermore, in the following detailed description, specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be recognized by an individual having ordinary skill in the art, i.e. a skilled person, that the present invention may be practiced without specific details, and/or with multiple details arising from combinations of aspects of particular embodiments. In a number of instances, known systems, methods, procedures, and components have not been described in detail so as to not unnecessarily obscure aspects of the embodiments of the present invention.
EXAMPLESA person skilled in the art will appreciate that the present invention may be practiced without undue experimentation according to the methods given herein. The methods, techniques and chemicals are as described in the references given or from protocols in standard biotechnology and molecular biology text books. Standard molecular biology techniques known in the art and not specifically described were generally followed as described in Sambrook and Russel, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2001).
Example 1: Materials and Methods for Fabrication of Protease-Responsive Particulate-Based Drug-Encapsulated Hybrid Hydrogels 1.1 Fabrication and Characterization of PLGA ParticlesParticles with or without ibuprofen were fabricated via an oil-in-water emulsification method with poly(lactic-co-glycolic acid) (PLGA) 50/50 (inherent viscosity of 0.95-1.20 dl/g) from Lactel (Pelham, Ala.) [Dang, T. T. et al. Biomaterials 34 (23), 5792-5801 (2013)]. Typically, a 5 mL solution of PLGA and ibuprofen dissolved in dichloromethane, at concentrations of 40 mg/ml and 6 mg/ml respectively, was quickly added to a 25 mL solution of 1% (w/v) polyvinyl alcohol (Sigma Aldrich, St. Louis, Mo., USA) and homogenized for 60 seconds at different speeds (L5M-A, Silverson). The resulting suspension was quickly decanted into 75 mL of deionized water and stirred for 60 seconds, followed by rotary evaporation for 15 minutes. The suspension was washed three times by centrifugation at 3000 rpm for 30 seconds. The particles were collected, flash-frozen in liquid nitrogen, and lyophilized to dryness. Particle size distribution and morphology were examined under a Scanning Electron Microscopy (JSM 6390LA, JEOL). The ibuprofen loading capacity of each microparticle formulation was determined by dissolving 2 mg of particles in 1 mL of acetonitrile and comparing the resulting UV absorbance at 240 nm to a standard curve of known concentrations of ibuprofen in acetonitrile. The release kinetics from the drug-loaded subdomain was independently investigated by varying the size of ibu-PLGA particles (Table 1 and
We developed a modular drug delivery platform which consisted of drug-loaded polymeric particles embedded inside a protease-cleavable hydrogel (
Peptide-crosslinked hydrogels were prepared by reacting 4-arm poly(ethylene glycol)-vinyl sulfone (PEG-VS) (20 kDa, Sigma Aldrich, St. Louis, Mo., USA) with bis-cysteine peptides (Genscript, Hong Kong) in stoichiometric ratio. Each precursor was dissolved in either triethanolamine (TEOA) buffer (0.3M) or PBS/NaOH buffer (pH=10). Typically, to prepare 117 μL of peptide-crosslinked hydrogels with 4.2% (w/v) PEG content, 5 mg of PEG-VS was dissolved in 100 μL of a buffer solution in a glass vial and mixed with a stoichiometric amount of a peptide crosslinker dissolved in 17 μL of the same buffer solution. Hydrogels with 4.2% (w/v) PEG content were evaluated in the preliminary screening of peptide crosslinkers. Hydrogels with 1.7% (w/v) PEG content were used in all subsequent in vitro and in vivo experiments. To form hybrid hydrogels consisting of PLGA particles (with or without ibuprofen) embedded in peptide-crosslinked hydrogels, the aforementioned precursors were dissolved separately in a buffer solution containing suspended PLGA particles at a concentration of 5% (w/v). By inverting the glass vial containing the liquid mixture of PEG-VS, a peptide crosslinker, and PLGA particles at regular intervals, gelation was confirmed when this liquid mixture did not flow downward despite gravity. Examples of peptide spacer sequences, peptide substrate sequences and protease susceptibility are shown in Table 2.
In this study, the peptide crosslinkers were designed with the ultimate objectives of forming hydrogels with PEG-VS and retaining its cleavability upon exposure to MMP-9 activity. Due to the modular design of the hybrid hydrogel, the peptide crosslinker, which is a key component of the subdomain determining MMP-9 cleavability, could be independently designed. Typically, a desired peptide crosslinker consisted of an MMP-9-cleavable substrate sequence flanked by two cysteine-containing spacer sequences, each of which comprised 4 amino acids. The substrates were selected from reported peptide sequences, which had been utilized as MMP-9-sensitive components in biosensors for MMP-9 detection or as MMP-9-cleavable linkers in drug-loaded nano-carriers for chemotherapy [Biela, A. et al. Biosensors and Bioelectronics 68, 660-667 (2015); Samuelson, L. E. et al. Molecular Pharmaceutics 10 (8), 3164-3174 (2013)]. The thiol moiety on the cysteine of each terminal spacer can be deprotonated to form a thiolate [Friedman, M. et al. Journal of the American Chemical Society 87 (16), 3672-3682 (1965)] which reacts with a vinyl sulfone moiety of PEG-VS via Michael addition reaction to induce gelation [Lutolf, M. P. and Hubbell., J. A. Biomacromolecules 4 (3), 713-722 (2003)].
To identify an optimal peptide crosslinker for GEL-iP, we performed qualitative screening of 8 peptide sequences (Table 3 and
Each hybrid hydrogel of 20 μL volume in a 500 μL Eppendorf tube was incubated at 37° C. with 3 μg/ml of MMP-9 (83 kDa, Merck) in PBS buffer (DPBS/modified, without calcium and magnesium, HyClone™). In a control experiment, another hybrid hydrogel with the same composition was immersed in PBS buffer without MMP-9. After 20 hours of incubation, the medium surrounding the hybrid hydrogel was sampled onto a cover slip and observed under an optical microscope (Olympus CKX53SF, Japan) to inspect for the presence of released particles.
The selected concentration of MMP-9 is within the range of MMP-9 expression in clinical wound fluids and synovial fluids of patients with rheumatoid arthritis and osteoarthritis [Ladwig, G. P. et al. Wound Repair and Regeneration 10(1) 26-37 (2002); Li, Z. et al. Journal of Diabetes and its Complications 27(4) 380-382 (2013)]. In a typical successful cleavage by MMP-9, the photographs of vials B1 and B2 in
In addition to substrate selection, spacer design (GCRR (SEQ ID NO: 9) or GCRD (SEQ ID NO: 8)) also plays an important role in the crosslinking process. For instance, in both buffers, spacer GCRR (SEQ ID NO: 9) helped peptide (2) crosslink with PEG-VS significantly faster than peptide (6) which was designed with the same substrate but a different spacer GCRD (SEQ ID NO: 8). Similarly, in PBS/NaOH, peptide (4) containing spacer GCRR (SEQ ID NO: 9) reacted with vinyl sulfones faster than peptide (5) which contained spacer GCRD (SEQ ID NO: 8). Our data was in agreement with published literature reporting that positive charges (e.g., arginine, R) close to thiol moieties of cysteines increased the crosslinking rate while negative charges (e.g., aspartic acid, D) decelerated this reaction [Lutolf, M. P. et al. Bioconjugate Chemistry 12 (6), 1051-1056 (2001)], possibly because the former stabilized the intermediate thiolates [Roos, G. et al. Antioxidants & Redox Signaling 18 (1), 94-127 (2012)].
Buffer choice can also affect the crosslinking rate because their environmental pH influences the deprotonation of thiols, leading to a change in the concentration of the intermediate thiolates [Lutolf, M. P. and Hubbell, J. A. Biomacromolecules 4 (3), 713-722 (2003)]. In addition to TEOA buffer, which is a strongly basic buffer commonly used for Michael addition but also raises cytotoxicity concern, we also investigated PBS/NaOH buffer as a potential cytocompatible alternative. As shown in
Next, the results in column (C) of
To design an effective protease-triggered drug delivery platform, an optimal peptide crosslinker should induce rapid gelation and retain its cleavability in response to this protease. Among the 8 screened peptides, 3 sequences (peptides (1), (4), and (7);
Two types of hybrid hydrogels were compared in this in vitro release study. GEL-iP was a hydrogel embedded with ibuprofen-loaded PLGA particles (ibu-PLGA particles) crosslinked with cleavable peptide (1) GCRR-KGPRSLSGK-RRCG (SEQ ID NO: 18). ScrGEL-iP was a hydrogel embedded with ibu-PLGA particles and crosslinked with non-cleavable scrambled peptide GCRR-KSSRGGPLK-RRCG (SEQ ID NO: 29). Briefly, 40 μL of GEL-iP was immersed in 500 μL of a PBS solution with or without 3 μg/ml of MMP-9 in a 1.5 mL tube while 40 μL of scrGEL-iP was only exposed to the MMP-9-containing PBS solution. In a control experiment with GEL-iP, MMP-9 inhibitor I (Merck) was added along with MMP-9. Each tube was maintained at a temperature of 37° C. and a shaking speed of 30 rpm on a Multi Bio RS-24 rotator (BioSan). At predetermined intervals, 10 μL aliquot of the liquid mixture from each tube was collected and added to 90 μL of acetonitrile. The resulting sample was passed through a 0.22 μm syringe filter and stored at 4° C. A 10 μL volume of fresh PBS solution with or without 3 μg/ml of MMP-9 was added to each tube to replace the aliquoted volume. After 24 hours, each hybrid hydrogel together with its remaining liquid mixture was completely dissolved in acetonitrile. The concentration of ibuprofen in all collected samples was quantified by RP-HPLC. The percentage of drug release at each time point was calculated by normalizing the cumulative amount of drug collected at each point with the initial amount of drug in each tube [Dang, T. T. et al. Biomaterials 32 (19), 4464-4470 (2011)]. The release kinetics reported for each hybrid hydrogel was obtained from the average of quadruplicate experiments.
As shown in
The drug released from GEL-iP upon exposure to MMP-9 trigger was evaluated by investigating its in vitro inhibitory effects on the proliferation of RAW 264.7 murine macrophages. Published studies previously demonstrated that local macrophage proliferation, rather than monocyte recruitment, dominates lesional accumulation of this cell type in inflammation-associated diseases such as atherosclerosis and obesity-associated adipose tissue inflammation [Amano, S. U. et al. (2014)]. Therefore, macrophage self-division has been postulated as a potential target for therapeutic modulation of inflammation.
RAW 264.7 murine macrophages were cultured in high glucose DMEM (Gibco Laboratories) supplemented with 10% FBS (Gibco Laboratories), and 1% penicillin/streptomycin (Gibco Laboratories) at 37° C. in 5% CO2 atmosphere. RAW 264.7 macrophages at passages of 20-30 were seeded in 96-well plates (Corning®) at an initial seeding density of 2×104 cells/well and incubated at 37° C. for 24 hours. A volume of 100 μL of each hybrid hydrogel (GEL-iP and scrGEL-iP) fabricated with 1.7% (w/v) of PEG and 5% (w/v) of ibuprofen-loaded PLGA microparticles was incubated in 500 μL of phenol red-free DMEM (Gibco Laboratories) culture medium for 2 hours in the presence and absence of 3 μg/ml of MMP-9 (
Relative metabolic activity=ATest/AControl*100%
Where ATest and AControl were the corrected absorbance values of solutions collected from the cells which were treated with the releasates and the fresh culture medium respectively.
As shown in
In addition, when MMP-9 inhibitor was added along with MMP-9, the metabolic activity of macrophages was restored to 20% from the complete inhibition (˜0%) observed in the absence of this inhibitor, proving that active MMP-9 is essential to achieve the desired inhibitory effects of releasates on macrophages. In the presence of MMP-9, while ibuprofen released from GEL-iP could completely inhibit macrophage proliferation, this activity still remained at 55% when the cells were treated with the releasate from non-cleavable scrGEL-iP. The findings from the control experiments with MMP-9 inhibitor and scrGEL-iP established the critical role of both active MMP-9 and its associated cleavable peptide in triggering ibuprofen release from Gel-iP to modulate macrophage proliferation. Overall, GEL-iP is a promising drug delivery platform which can be triggered by protease activity to release anti-inflammatory drugs and potentially modulate activity of immune cells.
While our main objective was to develop a delivery platform that releases drug only when triggered by protease activity with minimal release in the absence of this stimulus, some concerns arguably remain regarding the basal amount of ibuprofen released from GEL-iP in the absence of MMP-9. This may be due to passive drug diffusion from the surface of ibu-PLGA microparticles and accounted for the partial inhibition (˜40%) of macrophages treated with the releasate from Gel-iP in the absence of MMP-9 and its inhibitor. Nonetheless, in most actual clinical applications that require administration of an anti-inflammatory drug, some level of inflammation exists. Thus, this basal drug release can be useful for management of low level of inflammation and associated symptoms such as pain and swelling [Steinmeyer, J. (2000)] to minimize exacerbation of the inflammatory response [Sutherland, E. R. et al. (2003)]. If the inflammation suddenly worsens with a resultant increase in MMP-9 activity as in the case of an arthritic flare or infection of chronic wounds, GEL-iP will be triggered to release more ibuprofen to cope with the increased severity of inflammation.
Example 2: In Vivo Evaluation of Protease-Responsive Particulate-Based Drug-Encapsulated Hybrid Hydrogels 2.1 Animal Care of Immuno-Competent SKH-1 MiceTo further evaluate the potential use of GEL-iP as an injectable drug delivery platform for subcutaneous applications, we assessed the immuno-compatibility of GEL-iP and its constituent materials in vivo. An immunocompetent mouse model SKH-1E was utilized to investigate the effect of these materials on subcutaneous host response for up to 5 days (
Before subcutaneous injection of materials, mice were kept under inhaled anesthesia using 3% isoflurane in oxygen. Six different material formulations were subcutaneously injected in an array format on the dorsal side of each mouse. Specifically, a volume of 50 μL of PBS buffer containing ibu-PLGA particles (50 mg/ml), ibuprofen-free blank PLGA particles (50 mg/ml), or 1% (w/v) alginate hydrogel (PRONOVA™ SLG20, FMC BioPolymer) was injected. For each hydrogel formulation such as GEL-iP, GEL-P, or PEG hydrogel crosslinked by peptide (1) (GCRR-KGPRSLSGK-RRCG; SEQ ID NO: 18) without PLGA particles (PEG gel), 50 μL of a solution containing the corresponding precursors was injected. For example, in situ formation of GEL-iP was induced by subcutaneously injecting 50 μL of a PBS/NaOH buffer containing PEG-VS, the peptide crosslinker, and ibu-PLGA particles on the dorsal side of the mouse.
2.3 Non-Invasive Bioluminescent Imaging of SKH-1E MiceROS activity was quantified using luminol which was oxidized by ROS to emit bioluminescent signal as reported in other studies [Liu, W. F. et al. Biomaterials 32 (7), 1796-1801 (2011)]. Briefly, prior to imaging, the mice were injected with 5 mg of sodium luminol (Sigma Aldrich, St. Louis, Mo., USA) dissolved in 100 μL PBS into the peritoneum. Twenty minutes after this injection, the mice were imaged using the IVIS Spectrum CT system (Caliper Life Sciences) with 180 s exposure. Total flux (photons/s) was determined over a region of interest (ROI) (cm2) around the injection site using Living Image 3.1 software.
In a separate preliminary experiment, in situ gelation was confirmed by the presence of crosslinked hydrogels at the subcutaneous space of the excised skin 15 minutes post-injection (
Multiple in vitro and in vivo studies have quantified reactive oxygen species (ROS) activity generated by activated phagocytes to characterize material-induced host response [Dang, T. T. et al. Biomaterials 34 (23), 5792-5801 (2013); Dang, T. T. et al. Biomaterials 2011, 32 (19), 4464-4470 (2011)]. In this experiment, we used a non-invasive imaging technique to quantify bioluminescent signals emitted due to the oxidation of luminol imaging probe by ROS at the material injection site on days 1, 3, and 5 post-injection (
We demonstrated that particulate-based protease-cleavable hydrogels with plural responsivity were digested the fastest upon the coexistence of more than one disease-specific proteases, enhancing the specificity to target inflammatory diseases. Typically, the H2-M2 combinational hydrogels, which were crosslinked by a combination of a HNE peptide substrate (H2) and MMP-9 peptide substrate (M2) (Table 4), remained intact with the exposure to only a single protease, either HNE or MMP-9. However, the hydrogels were fully degraded when both proteases were added (
To illustrate the versatility of this drug delivery platform for topical applications, we incorporated the hybrid hydrogel GEL-iP with Kaltostat® wound dressing to form a composite dressing (
Since the newly-formed composite dressing was saturated with the water from the precursor mixture, we observed that its ability to further absorb liquid significantly decreased. Thus, the composite dressing was lyophilized to restore its absorbability. This dressing was then investigated for its ability to release ibuprofen upon exposure to MMP-9 by immersing it in a buffer solution with or without MMP-9 for 24 hours. After 24-hour incubation, the composite dressing rapidly absorbed the buffer and released nearly 100% of loaded ibuprofen in the presence of MMP-9 compared to only 56% in the absence of MMP-9 (
We have developed a robust process to synthesize and purify the new ibuprofen-peptide conjugate. The process flowchart is shown in
First, ibuprofen, a non-steroid anti-inflammatory drug (NSAID), was conjugated to the N-terminus of a peptide sequence, GPQGIWGQ-DRCG (SEQ ID NO: 19), to form ibu-GPQGIWGQ-DRCG (SEQ ID NO: 19) using solid phase peptide synthesis (SPPS). Fmoc-protected peptide was first synthesized on 0.3 mmol/g scale on Rink amide resin using standard manual solid phase peptide synthesis. Prior to ibuprofen conjugation, the Fmoc protection group was removed with 20% piperidine. An amount of 50 mg of the Fmoc-free resin was then dispersed in 500 μL of DMF along with 9.28 mg of ibuprofen. The reaction occurred when 34.2 μL of 1M PyBOP solution in DMF and 6 μL of DIPEA were added into the resin dispersion. After 18 hours, the resin was washed with DMF then dichloromethane (DCM) several times. Next, ibu-GPQGIWGQ-DRCG (SEQ ID NO: 19) was cleaved off the resin using a cleavage cocktail containing 95% trifluoroacetic acid, 2.5% water and 2.5% triisopropylsilane (TIPS) for 60 min at room temperature. The product was precipitated in cold ether, then kept under vacuum to dryness. The identity of the peptide-drug conjugation was confirmed with MS, MS m/z: 731.35 [M+2H]2+. The LC-MS data indicated that the drug has been successfully conjugated to the N-terminus of the peptide sequence, GPQGIWGQ-DRCG (SEQ ID NO: 19), as the observed molecular weight of ibu-GPQGIWGQ-DRCG (SEQ ID NO: 19) matched the theoretically predicted values from ChemDraw® (
Second, ibu-GPQGIWGQ-DRCG (SEQ ID NO: 19) was linked to the hydrogel using the following procedure. 4-arm poly(ethylene glycol)-maleimide (4-PEG-Mal) (20 kDa, Sigma Aldrich, St. Louis, Mo., USA) first reacted to ibu-GPQGIWGQ-DRCG (SEQ ID NO: 19) with a mole ratio of 1/1. Bis-cysteine peptides (Genscript, Hong Kong), in stoichiometric ratio to 4-PEG-Mal (albeit the amount of maleimide groups occupied by ibu-GPQGIWGQ-DRCG (SEQ ID NO: 19)) was then added to the reaction mixture. Each precursor was dissolved in PBS buffer. Typically, to prepare 117 μL of peptide-crosslinked hydrogels with 4.2% (w/v) PEG content, 5 mg of 4-PEG-Mal was dissolved in 100 μL of PBS along with 0.40 mg of ibu-GPQGIWGQ-DRCG (SEQ ID NO: 19) in a glass vial. Next, a stoichiometric amount of a peptide crosslinker dissolved in 17 μL of PBS was added to the solution.
As demonstrated by the schematics in
Third, preparation of plural protease-cleavable hydrogels was accomplished through Michael-type addition reaction of thiol-containing peptides onto 4-PEG-Mal. A gel of 117 μL volume containing 4.2% (w/v) 4-PEG-Mal was formed by dissolving 5 mg 4-PEG-Mal in 100 μL PBS buffer and reacting this solution with 17 μL of a combination of peptide sequences, such as 0.005 mg of GRCR-PMAVVQSVP-RCRG (SEQ ID NO: 31) and 0.0045 mg of GRCR-GPRSLSG-RCRG (SEQ ID NO: 32).
5.2 Tunable In Vitro Drug Release from Conjugate-Based HydrogelQuantitative data determined from high performance liquid chromatography (HPLC) as shown in
We also obtained preliminary data suggesting the drug release from our drug-conjugated platform was able to release higher drug dose in response to increased inflammation in vivo. We established a mouse model of skin inflammation using phorbol 12-myristate 13-acetate (PMA) as a stimulant (
To investigate the in vivo inflammation-triggered release of drug from the ibuprofen-conjugated PEG hydrogels, ibu-M_xM hydrogel was formed in situ by injecting its precursor solution in the subcutaneous space of the inflamed sites on the dorsal side of SKH-1E mice (
All statistical analysis and graphing were processed with OriginPro 2017. All comparisons between two experimental groups were determined using two tailed Welch's t-test, while those between more than two groups were done using one-way ANOVA analysis with Fisher LSD post-hoc test. P-values less than 0.05 were considered significant.
Example 6: Plural Protease-Triggered In Vitro Drug Released from Modular Conjugate-Based HydrogelTypically, we used the following procedure to prepare 20 μL of dual protease-triggered modular conjugate-based hydrogel. Firstly, 10 mg of 8-arm PEG-maleimide (8-PEG-MAL, 40 kDa) was dissolved in 98.33 μL of a PBS buffer solution in a 0.5 ml Eppendorf tube. 0.17 mg MMP-9 sensitive peptide (GPRSLSG-RRCG; SEQ ID NO: 20) linked ibuprofen (Mibu, 1332.7 mmol/mg) was dissolved in 1.67 μL of dimethyl sulfoxide (DMSO) and mixed with the 8-PEG-MAL solution in a stoichiometric ratio of 1:2. After that, 1.62 mg of MMP-9 degradable peptide crosslinker (GCRR-GPRSLSG-RRCG; SEQ ID NO: 21) and 1.87 mg HNE degradable peptide crosslinker (GRCR-PMAVVQSVP-RCRG; SEQ ID NO: 31) were dissolved in 17 μL of the PBS buffer solution separately and mixed in a stoichiometric ratio of 1:1. Final PEG-peptide hydrogels were prepared by reacting the mixture of 8-PEG-MAL and Mibu with the mixture of two peptide crosslinkers in a stoichiometric ratio of 4:1. The final hydrogel solutions were homogenized by vortexing and centrifugation for 3 seconds respectively. Crosslinking time was counted by observation, starting from initial mixing time until there was no free flow solution observed. The hydrogels were also flicked and observed under room light. Formation of the hydrogels were confirmed when transparent and intact hydrogels were observed stick at bottom of the tube and without forming any bubble inside when hydrogel tubes were flicked vigorously. Hydrogels then were used in all subsequent in-vitro gel degradation and drug release experiments.
Briefly, 20 μl of PEG-peptide hydrogels were immersed in 200 μl of PBS solution with or without an enzyme mixture MMP-9 (2 μg/ml) and HNE (1 μg/ml) in a 1.5 mL tube respectively while another two 20 μl of PEG-peptide hydrogels were exposed to the MMP-9-containing HEPES solution (2 μg/ml) or HNE-containing HEPES solution (1 μg/ml) respectively. Each tube was incubated at 37° C. At predetermined time intervals (4, 8, 12, 24, 36, 48 hours), 5 μl aliquot of the liquid sample from each tube was collected and added to 15 μl of buffer solution to dilute it four times in a 2 ml glass vial. A 5 μl volume of the corresponding buffer solution was added to each tube to replace the aliquoted volume. The concentration of digested peptide fragments in all collected samples was quantified by HPLC. The HPLC analysis was conducted at room temperature. The mobile phase used in HPLC was ultra-pure water and acetonitrile at a volume ratio of 35/65 with 0.1% trifluoroacetic acid at a flow rate of 1.0 mL/min. The detected spectrum was converted to drug concentration, which was used to calculate the cumulative percentage of drug release at predetermined time points. The calculation was done by dividing the cumulative amount of drug release (sum of instantaneous drug release and drug loss at previous time points) by the initial amount of peptides used in each hydrogel. The release profile was generated by plotting cumulative percentage drug release versus release time. Data at each time point was obtained from the average of triplicate experiments with a standard deviation.
Release kinetics of an anti-inflammatory drug from the hydrogel was investigated in vitro in response to the presence of enzyme solution containing MMP-9 or HNE or MMP-9/HNE mixture. The control experiment was done in pure buffer solution without enzyme. The release profile was obtained by plotting cumulative release at predetermined time points (4, 8, 12, 24, 36, 48 hours after exposure to enzyme solutions), shown in
For administration of anti-inflammatory therapeutics, multiple strategies have been attempted to improve spatiotemporal control of drug release kinetics including physically encapsulating drugs or permanently conjugating drugs to polymer backbones [14-17]. However, the release kinetics of these systems could not adapt to changes in the severities of inflammatory diseases. Exploiting enzymes upregulated during arthritic flares as biological cues to activate the drug release, Joshi et al. leveraged the self-assembly of triglycerol monostearate (TG-18) to physically entrap a corticosteroid in a hydrogel platform [20]. However, the drug release from this platform relies on the cleavage of ester bonds on the TG-18 backbone primarily by esterases. Non-enzymatic hydrolysis of these ester bonds in the low pH environment associated with inflammatory conditions [24-26] might also result in undesirable non-specific drug release.
Our invention focused on designing better-performing drug delivery platform with improved control over basal release rate and/or enhanced selectivity and specificity to the inflammation-associated condition. Overall, we have demonstrated several advantageous characteristics of this platform: (1) modular system design consisting of multiple integrating subdomains, each of which possessed a distinct function and could be created and replaced individually to tailor drug loading and drug release kinetics to specific inflammation-associated conditions/diseases by varying the chemical composition of constituent material; (2) the ability to tailor the basal release rate by either significantly minimizing the basal release of drug via covalent conjugation of drugs/modified drugs to the inflammation-responsive hydrogels through protease-cleavable peptides or maintaining some moderate basal release using drug-loaded polymeric particles as the drug-containing domain; (3) the combination of peptide sequences to enable the platform to release loaded cargo upon exposure to one or more disease-specific protease(s), potentially enhancing its specificity to release tailored dosage correlating with the inflammation severity of the disease. These advantages are demonstrated in several examples below.
Modular Particulate-Based Hydrogel with Singular or Plural Protease Responsivity
We developed a modular hybrid hydrogel which could be triggered to release an anti-inflammatory drug upon exposure to elevated protease activity associated with inflammatory diseases. Upon exposure to protease activity, the hydrogel matrix could be proteolytically degraded to liberate the embedded particles and consequently deliver the desired therapeutic payload. Modular design of the hybrid hydrogel enabled independent optimization of its protease-cleavable and drug-loaded subdomains to facilitate hydrogel formation, cleavability by matrix-metalloprotease-9 (MMP-9) to ultimately deliver desirable payload at tunable release rate. In vitro study demonstrated the protease-triggered enhancement of drug release from the hybrid hydrogel system for effective inhibition of TNF-α production by pro-inflammatory macrophages and suggested its potential to mitigate drug-induced cytotoxicity. Using non-invasive imaging to monitor the activity of reactive oxygen species in biomaterial-induced host response, we confirmed that the hybrid hydrogel and its constituent materials did not induce adverse immune response after 5 days following their subcutaneous injection in immuno-competent mice. We subsequently incorporated this hybrid hydrogel onto a commercial wound dressing which could release the drug upon exposure to MMP-9. Together, our findings suggested that this hybrid hydrogel might be a versatile platform for on-demand drug delivery via either injectable or topical application to modulate inflammation in chronic diseases.
Modular Conjugate-Based Hydrogel with Singular or Plural Protease Responsivity
Modular hydrogel systems conjugated with anti-inflammatory drug was also developed. We demonstrated that triggered release of therapeutic drug can be achieved by singular or dual protease stimuli. In some embodiments, the drug loading capacity of the drug-conjugated hydrogel system could be increased by manipulating the configuration of polyethylene glycol which was the hydrogel backbone. The drug release rate was tuned by changing protease-cleavable peptide anchors and crosslinkers. In addition, the in vivo protease-triggered drug release was demonstrated using a model of chemically induced subcutaneous inflammation with different severity levels.
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Claims
1. A drug-loaded protease-responsive hydrogel comprising; wherein said polymer building block of b) forms a gel in the presence of the protease-cleavable crosslinker of c) to entrap the particles of a).
- a) a drug encapsulated in particles;
- b) a polymer building block comprising of a multi-arm-polyethylene glycol (PEG) with functional moiety; and
- c) a bis-functional protease-sensitive crosslinker comprising a protease-cleavable substrate flanked by two spacer sequences containing functional moieties;
2. The drug-loaded protease-responsive hydrogel of claim 1, further comprising:
- a) at least a second bis-functional protease-sensitive crosslinker which comprises a protease-cleavable substrate, sensitive to a different protease to that of said crosslinker of c), flanked by spacer sequences containing functional moieties; and/or
- b) at least a further bis-functional protease-resistant crosslinker which comprises a protease-resistant substrate.
3. The drug-loaded protease-responsive hydrogel of claim 1 or 2, wherein the drug is encapsulated in particles comprising a material selected from the group comprising silica, liposomes, siRNA complexes and polymeric materials such as polycaprolactone, poly (methacrylic acids), polylactic acids, polyvinylpirrolidone, poly(lactic-co-glycolic acid) (PLGA), and gelatin.
4. The drug-loaded protease-responsive hydrogel of any one of claims 1 to 3, wherein the polymer building block comprises a multi-arm-PEG-vinyl sulfone or multi-arm-PEG-maleimide or multi-arm-PEG-azide or multi-arm-PEG-alkyne.
5. A drug-loaded protease-responsive hydrogel comprising; wherein the functional moiety of the peptide anchor covalently links the drug to an arm of the multi-arm PEG polymer, and wherein a functional moiety of said polymer building block covalently links to said moiety of said bis-functional crosslinker to form a gel.
- a) a drug covalently conjugated to a protease-cleavable peptide anchor having a functional moiety;
- b) a polymer building block comprising a multi-arm-PEG polymer having at least one functional moiety; and
- c) a bis-functional crosslinker comprising a peptide substrate flanked by spacer sequences containing functional moieties;
6. The drug-loaded protease-responsive hydrogel of claim 5, wherein:
- a) said crosslinker is not cleavable to a protease; or
- b) said peptide anchor is cleavable to a protease and said crosslinker is cleavable to the same or different protease and/or
- c) said drug-loaded hydrogel comprises a plurality of crosslinkers, one or more of which are cleavable to different proteases.
7. The drug-loaded protease-responsive hydrogel of claim 5 or 6, wherein the polymer building block comprises a multi-arm-PEG-vinyl sulfone or a multi-arm-PEG-vinyl Maleimide or a multi-arm-PEG-azide or a multi-arm-PEG-alkyne.
8. The drug-loaded protease-responsive hydrogel of claim 7, comprising 2-12 wt % multi-arm-PEG-vinyl Maleimide.
9. The drug-loaded protease-responsive hydrogel of any one of claims 1 to 8, wherein the multi-arm PEG polymer has 3 to 8 arms.
10. The drug-loaded protease-responsive hydrogel of any one of claims 1 to 9, wherein the drug is anti-inflammatory.
11. The drug-loaded protease-responsive hydrogel of claim 10, wherein the drug is a non-steroidal anti-inflammatory drug (NSAID).
12. The drug-loaded protease-responsive hydrogel of claim 10 or 11, wherein the protease is upregulated during inflammation and is selected from the group comprising matrix metalloproteinases, serine proteases, cysteine proteases and aspartic proteases.
13. The drug-loaded protease-responsive hydrogel of any one of claims 1 to 12, wherein the said flanking spacer sequences comprise at least one Cysteine and/or Lysine residue and/or azide- or alkyne-containing unnatural amino acid.
14. The drug-loaded protease-responsive hydrogel of claim 13, wherein the said flanking spacer sequences comprises a sequence of 1-6 amino acids.
15. The drug-loaded protease-responsive hydrogel of any one of claims 1 to 14, wherein the protease-cleavable substrate is sensitive to a protease selected from the group comprising matrix metalloproteinase, such as metalloproteinase-9 (MMP-9), MMP-2, MMP-7, MMP-12 etc., cathepsins, such as Cathepsin K, Cathepsin B, Cathepsin S, etc., and human neutrophil elastase (HNE), caspases and urokinases.
16. The drug-loaded protease-responsive hydrogel of claim 15, wherein the protease-cleavable substrate is selected from the group comprising MMP-9 substrates comprising the amino acid sequence set forth in KGPRSLSGK (SEQ ID NO: 30), GPRSLSG (SEQ ID NO: 10), LGRMGLPGK (SEQ ID NO: 11), AVRWLLTA (SEQ ID NO: 12) or GPQGIWGQ (SEQ ID NO: 13; HNE substrates comprising APEEIMDRQ (SEQ ID NO: 14) or PMAVVQSVP (SEQ ID NO: 15; Cathepsin B substrates comprising GRRGLG (SEQ ID NO: 16) or DGFLGDD (SEQ ID NO: 17) or a combination thereof.
17. A composition comprising the drug-loaded protease-responsive hydrogel of any one of claims 1 to 16 formulated for injection or topical administration.
18. A dressing comprising the drug-loaded protease-responsive hydrogel of any one of claims 1 to 16.
19. Use of the drug-loaded protease-responsive hydrogel of any one of claims 1 to 16 or a composition of claim 17 as an injectable or topical dressing for treating a subject in need thereof.
20. A method of treatment comprising administering to a subject in need of such treatment an efficacious amount of the drug-loaded protease-responsive hydrogel of any one of claims 1 to 16 or a composition of claim 17.
21. A kit comprising:
- a) a drug encapsulated in particles;
- b) a polymer building block comprising of a multi-arm-polyethylene glycol (PEG) with functional moiety; and
- c) a bis-functional protease-sensitive crosslinker comprising a protease-cleavable substrate flanked by two spacer sequences containing functional moieties,
- wherein a)-c) are as defined in any one of the previous claims; or
- a) a drug covalently conjugated to a protease-cleavable peptide anchor having a functional moiety;
- b) a polymer building block comprising a multi-arm-PEG polymer having at least one functional moiety; and
- c) a bis-functional crosslinker comprising a peptide substrate flanked by spacer sequences containing functional moieties,
- wherein a)-c) are as defined in any one of the previous claims.
22. A method of manufacturing a drug-loaded protease-responsive hydrogel comprising the steps: wherein said polymer building block of a) forms a gel in the presence of the protease-cleavable crosslinker of b) to entrap the drug-loaded particles.
- a) mixing a polymer building block comprising of a multi-arm-polyethylene glycol (PEG) with functional moiety with drug-loaded particles;
- b) mixing a bis-functional protease-sensitive crosslinker comprising a protease-cleavable substrate flanked by spacer sequences containing functional moieties with drug-loaded particles;
- c) mixing together the mixtures of a) and b);
23. A method of manufacturing a drug-loaded protease-responsive hydrogel comprising the steps: wherein a functional moiety of said polymer building block covalently links to said moiety of said bis-functional crosslinker to form a gel.
- a) mixing a drug covalently conjugated to a peptide anchor having a functional moiety with a polymer building block comprising a multi-arm-PEG polymer having at least one functional moiety, wherein the respective functional moieties of the peptide anchor and multi-arm-PEG polymer covalently bond to conjugate the drug to an arm of the multi-arm PEG polymer;
- b) mixing the drug-polymer conjugate of a) with a bis-functional crosslinker comprising a peptide substrate flanked by spacer sequences containing functional moieties;
24. The method of claim 23, wherein:
- a) said peptide anchor is cleavable to a protease and said crosslinker is not cleavable to a protease; or
- b) said peptide anchor is cleavable to a protease and said crosslinker is cleavable to the same or different protease; and/or
- c) said drug-loaded hydrogel comprises a plurality of crosslinkers, one or more of which are cleavable to different proteases.
25. The method of any one of claims 22 to 24, wherein the drug, the polymeric particles, the crosslinker, the cleavable anchor and/or the polymer building block are as defined in any one of claims 1 to 16.
26. A method of manufacturing a composite dressing comprising a drug-loaded protease-responsive hydrogel of any one of claims 1 to 16, comprising the steps;
- a) preparing a mixture of a drug-encapsulated in particles and a bis-functional protease-sensitive crosslinker comprising a protease-cleavable substrate flanked by two spacer sequences containing functional moieties;
- b) preparing a mixture of a drug-encapsulated in particles and a polymer building block comprising of a multi-arm-polyethylene glycol (PEG) with functional moiety;
- c) mixing a) and b) together, depositing the mixture onto a dressing and allowing gelation.
27. The method of claim 26, wherein the dressing is an alginate wound dressing.
28. The method of claim 26 or 27 further comprising step d), wherein the composite dressing is flash-frozen in liquid nitrogen and lyophilized to dryness.
29. The method of any one of claims 22 to 28, wherein the drug is a NSAID; the particle comprises poly(lactic-co-glycolic acid) (PLGA); the crosslinker and/or anchor are cleavable to a protease selected from the group comprising matrix metalloproteinases and serine proteases or combinations thereof; and the polymer building block comprises a 4- or 8-arm-PEG-vinyl sulfone or a 4- or 8-arm-PEG-vinyl Maleimide or a 4- or 8-arm-PEG-azide or a 4- or 8-arm-PEG-alkyne.
Type: Application
Filed: Dec 7, 2020
Publication Date: Feb 9, 2023
Inventors: Thuy Tram DANG (Singapore), Tri Dang NGUYEN (Singapore), Hsin-Yueh NG (Singapore)
Application Number: 17/782,908